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Horizontal gene transfer and the
origin of species: lessons from bacteria
Fernando de la Cruz and Julian Davies
T
he old topic of horizonIn bacteria, horizontal gene transfer
help to explain such a broadtal gene transfer (HGT)
(HGT) is widely recognized as the
host-range process (i.e. one
has become fashionable1. mechanism responsible for the widespread that operates not only between
From the overwhelming surge distribution of antibiotic resistance genes, species and genera, but also
of genome sequence inforgene clusters encoding biodegradative
between kingdoms). In this
mation, more and more candi- pathways and pathogenicity determinants. particular case it is apparent
dates for horizontally trans- We propose that HGT is also responsible that, once again, we can learn
from our vast experience with
ferred genes are being identified:
for speciation and sub-speciation in
prokaryotes, especially as typical
among prokaryotes2; from
bacteria, and that HGT mechanisms
methods of carrying out HGT
bacteria to eukaryotes3; from
exist in eukaryotes.
in prokaryotes, such as transbacteria to archaea4; from aniF. de la Cruz* is in the Departamento de Biología
fection or even bacterial conjumals to bacteria5; and so on. It
(Unidad Asociada al C.I.B.), Universidad
gation6,7, can be used for the
is clear that genes have flowed Molecular
de Cantabria, Santander, Spain; J. Davies is in the
through the biosphere, as in a
genetic manipulation of eukaryDept of Microbiology and Immunology,
global organism. HGT, once
otic cells. There are a number
University of British Columbia, Vancouver, BC,
Canada V6T 1Z3.
solely of interest for practical
of well documented examples
*tel: +34 942 201942,
applications in classical genin which bacterial adaptation
fax: +34 942 201945,
etics and biotechnology, has
has been influenced by HGT.
e-mail: [email protected]
now become the substance of
Studying these examples can
evolution. However, amid this
provide an appreciation of the
new-found enthusiasm for an old subject, the existence general mechanistic trends involved, as well as providof HGT in eukaryotes is viewed with skepticism, as ing the foundation for considerations of a ubiquitous
there is a distinct lack of mechanistic studies that would role of HGT in shaping modern eukaryotic species.
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
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Table 1. Mobile genetic elements involved in HGT in bacteria
Mobile element
General features
Examples
Phenotype of specific element
Ref.
Gene cassette
Mobility of ‘simple genes’
Immediate constitutive expression
Extremely broad host range
IntI1
IntI4
Antibiotic resistance (simple)
Gene ‘maturation’ in Vibrio
30
31
Transposon
Mobility of genes and operons
Very broad host range
Loci screening for best performance
Tn21
Tn4651
Antibiotic resistance (complex)
Toluene degradation
32
33
Plasmid
Mobility of operons and regulons
Immediate regulated expression
Very broad host range
pNRG234a
pNL1
pCD1
Nodulation plasmid of Rhizobium
Degradation of aromatic compounds
Virulence determinants of Yersinia
34
12
35
Bacteriophage
Mobility of regulons
‘Maturation’ in chromosomes
(via lysogeny)
Narrow host range
CTXF
Virulence phage of Vibrio cholerae
36
Abbreviation: HGT, horizontal gene transfer.
Antibiotic resistance: the immediate response
The dissemination of antibiotic resistance genes
among human and animal bacterial pathogens and
associated commensal populations is the paradigm
for HGT on a global scale8. This ‘experiment’ has
taken place during the past 50 years, and a significant
amount of information has been accrued on the
mechanisms of response (evolution) of different bacterial ecosystems to the addition and distribution of
large amounts of toxic agents (antibiotics) within the
biosphere. This is the best known example of very
rapid adaptation of bacterial populations to a strong
selective pressure. What has been learned is that this
adaptation occurred (and still occurs if bacteria are
challenged with a novel antibiotic) not by mutation of
the menaced populations, but by acquisition and dissemination of, in most cases, simple antibiotic resistance genes by mobile genetic elements (Table 1). It
should be noted that studies of antibiotic resistance
development are essentially retrospective, and although much has been learned, little is known of the
microbial dynamics of this process. Nonetheless, the
basis of antibiotic resistance development is formally
simple: mobile genetic elements such as plasmids and
transposons efficiently distributed the resistance determinants, singly or in clusters, among many genera
and species of bacteria. Intergeneric boundaries were
not respected, particularly the long-standing but
only apparent distinction between Gram-positive and
Gram-negative bacteria. This latter distinction is almost certainly a figment of human imagination that
has been ignored by microorganisms for billions of
years.
Biodegradation pathways: delayed opportunistic
response
Based on studies with antibiotic resistance and its associated genes (e.g. genes for the metabolism of a
number of sugars), it is not surprising that HGT has
also led to the dissemination of gene clusters (operons) involved in the catabolism of xenobiotics in
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polluted environments9–11. The selective pressures are
similar to those of antibiotic resistance, but the
process of degrading xenobiotic compounds requires
more complex genetic systems, usually operons of ten
or more genes, or even regulons of several operons
with accompanying control circuits. An example of
this can be found in the Sphingomonas aromaticovorans plasmid pNL1 (Ref. 12). This large conjugative
plasmid contains 15 gene clusters directly associated
with the catabolism or transport of aromatic compounds, allowing the host bacteria to degrade compounds such as biphenyl, naphthalene, xylene and
cresol. Although the dissemination of biodegradation
pathways has not been analysed to the same extent as
that of antibiotic resistance, the available data clearly
indicate that the genetic mechanisms responsible are
likely to have been the same: plasmids and transposons distributed catabolic operons among a wide
range of bacteria (see Ref. 13). It can be assumed that
the degradation of xenobiotics by bacteria has intensified in the past century, as a result of the increased
use of chemicals in industry and the waste disposal
habits of humans, but the basic processes could have
evolved much earlier. For example, pathways for the
degradation of recalcitrant aromatic compounds resemble lignin degradation pathways, from which they
probably derive14. This is in contrast to antibiotics,
which were not in use before the 1940s (with the exception of sulfonamides, disinfectants and mercury
compounds), a time when antibiotic resistance genes
were very scarce among human pathogenic bacteria15,16.
Significantly, degradative operons are also found integrated in the bacterial chromosome. In these cases,
adjacent remnants of phage or plasmid genes remind
us of the once-mobile nature of these operons.
Pathogenicity determinants: delayed internalized
response
Judging from these examples, it would appear that
plasmids, in combination with transposons, are the
primary vehicles for HGT in bacteria, at least for
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emergency responses to strong selection pressures.
What other traits do naturally occurring plasmids
and transposons carry? The answers to this question
could give clues about bacterial responses to other
types of stress, and perhaps help discover other evolutionary forces that shaped bacterial species. Plasmids in pathogenic bacteria are loaded with virulence
determinants, ranging from toxin production and immune evasion, to overt sabotage of the cellular machinery, as in the case of the oncogenic Ti plasmid.
The importance of ‘pathogenic’ plasmids cannot be
overemphasized as, in many cases, they carry the
backbone of genetic information that makes a given
bacteria a pathogen, so much so that they can be considered speciation determinants. The Ti plasmids of
Agrobacterium, the nodulation plasmids of Rhizobium and the virulence plasmids of Yersinia, Shigella
and Escherichia coli are examples of plasmids that, in
essence, create new bacterial species when they are
acquired, so drastic are the phenotypic variations
they beget. Thus, virulence, that is the mechanisms of
bacterial adaptation that allow the colonization of a
new niche when that niche is created by the appearance of a new multicellular organism, occurs not
by mutation, but by the acquisition of pathogenicity
determinants by HGT.
However, not all pathogenic determinants are
found on plasmids. One of the first recognized examples of a virulence gene was that encoding the toxin of
Corynebacterium diphtheriae, the agent of diphtheria,
which is carried by a temperate phage (a dormant
phage, integrated into the host chromosome). Numerous other cases of temperate phages carrying
virulence determinants have been identified in a range
of bacterial pathogens, such as Pseudomonas aeruginosa, Vibrio cholerae, Shigella dysenteriae and
E. coli17. Whatever the frequency of pathogenicity
plasmids or phages, genomic studies have shown that
most pathogenicity genes are located in the bacterial
chromosome, where they exist as discrete gene clusters named pathogenicity islands. It would appear
that pathogenicity can be considered a new epitome
in the evolution of bacterial species.
The ability to survive in very specific hostile environments, such as on and within other organisms, can
be assumed to be a slow evolutionary process compared with the occurrence of resistance determinants.
Pathogenicity islands are the evolutionary paradigm
of slow selection pressure. They are mostly integrated
into their host bacterial chromosomes, but evidence
suggests that they are, or were, mobile elements. In
most cases, they resemble defective bacteriophages,
although occasionally they are associated with the
remnants of plasmid conjugative systems or compound transposons17. Bacteriophages are probably
effective tools by which HGT can provide a more permanent adaptation to new environments. One or
more regions of DNA are acquired by fortuitous
transduction, by their inclusion in defective phage or
by Hfr-type conjugation in the case of the donor
being a plasmid, and incorporated into the bacterial
chromosome. Subsequently, the bacteriophage or
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plasmid vehicles are rendered inactive by deletion or
insertional inactivation, so that the incorporated
genes are fixed. On the evolutionary scale, the acquisition of pathogenicity islands occurs at a much
slower pace than that of plasmids, and would obviously create new bacterial subspecies (i.e. the recombinants have discernibly different phenotypes). Because multiple pathogenicity islands, varying in size
and in location on the host chromosome, can be
found in any bacterial strain, they must have been inherited independently and could well have come from
different donor organisms at different times via different routes. It is not yet known if pathogenicity islands go through a process of being ‘hosted’ and then
adapted to different recipient bacterial genera and
species before their identification in a contemporary
clinical isolate. This introduces a pertinent concept:
that of quantum evolutionary leaps. Pathogenicity islands probably did not arrive in their contemporary
host as the result of a single event; rather, they were
hosted and adapted to one or more intermediate
species on their path to identification.
The shaping of a bacterial species
The fact that a number of pathogenicity determinants
are presently found on mobile elements, or, alternatively, are fixed in the chromosome but still show
remnants of the vehicles that led them there, suggests
that chromosomes, classically viewed as essentially
immobile, are in fact the ‘genetic necropolis’ of previously mobile genes. Who knows the full extent of
micro-geographical terms that will spring from bacterial genomics: we could soon have islets, peninsulas
or even genetic archipelagos! Armed with the awareness that HGT is so important in bacterial speciation,
it is now possible to examine completely sequenced
chromosomes in a new light and assess the extent of
chromosomal DNAs of obscure lineage.
Using E. coli and Salmonella as a model system, it
has been shown that 17% of their genomes (i.e.
~800 kb), appears to have been acquired by HGT
during the past 100 million years (the housekeeping
genes of both organisms are ~90% identical)18. As the
majority of this DNA was recruited recently, it is apparent that considerable genetic flux is occurring: the
234 detectable HGT events that have persisted are
probably the tip of the iceberg of the thousands of
mobile sequences that have entered and left any particular E. coli strain. Similarly, when the members of
a well known collection of E. coli strains (the ECOR
collection) were compared, they were found to be
quite variable in the size and macro-organization of
their chromosomes19,20 and plasmids21,22. Additionally, a significant proportion of the Salmonella ‘unique’
genes are involved in virulence, underlying the importance of HGT-acquired genes for speciation23. In
summary, a significant proportion of the genome of
any strain of a single bacterial species comprises fragments of DNA from various origins which, properly
‘nurtured’, can give rise to new bacterial species.
How many HGT steps are involved in the creation
of a new species? We can use E. coli O157:H7 as an
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example of a new ‘quasi-species’. AlTable 2. Pathogenicity-related mobile elements of Escherichia coli
though its genome has not been com157:H7 acquired by HGT
pletely sequenced, it is known to contain more than 20 potential virulence
Name
Type
Size (kb)
Virulence factors
Ref.
genes clustered in several mobile elements: the large plasmid pO157, the
pO157
Plasmid
92
Hemolysin
37
lambdoid temperate phage 933W,
Large clostridial toxin
another cryptic prophage and the
Catalase–peroxidase
pathogenicity island LEE, all textExtracellular serine protease
book examples of modular construc933W
Lysogenic phage
62
Shiga toxin 2
38
tion of pathogenicity determinants
Ser/Thr protein kinase
by HGT (Table 2). The genome of
Three novel tRNA genes
E. coli O157:H7 undoubtedly conMacrophage survival
tains more surprises, as it is 700 kb
Serum resistance
larger than that of E. coli K-12. GenLEE
Pathogenicity island 43
Intimin
39
omic comparisons of a number of
Tir
E. coli strains, including pathogenic
variants, will define the essential core
Abbreviations: HGT, horizontal gene transfer; Tir, translocated intimin receptor.
of the E. coli chromosome, and will
give a more significant idea of the
collective amount of mobile DNA available as an or loss of specific sets of genes as a result of HGT, speextended genotype, including the inserts that made ciation by HGT is not a specific response to defined
E. coli O157:H7 highly pathogenic for humans. We challenges, but a global evolutionary response of bacwould like to contrast the immediate and powerful terial populations. The E. coli scenarios already disHGT events that resulted in E. coli O157:H7 with the cussed provide evidence for this. Why should this not
results obtained by ‘solitary confinement’ evolution be the case also in eukaryotes and why should they
of E. coli in the laboratory during 20 000 gener- not use the same or very similar mechanisms, being
ations24, where the bacterial genome remains basically available as they are in prokaryotes? We propose that
intact if allowed to evolve (presumably to a simpler major evolutionary leaps in eukaryotes (most clearly
genome adapted to rapid growth in a predictable non- in unicellular eukaryotes, but possibly also in multivariant medium) without the help of acquired exoge- cellular organisms) are produced by the traffic of monous DNA. It will be exciting to compare these results bile elements that operate in the same way as bacwith an experimental set-up in which additional bac- terial mobile elements. The eukaryotic mobile genetic
terial DNA is allowed, periodically, to enter the system. elements are the transposons, viruses and bacteria
It would therefore appear that much of the specia- that thrive among them. Initial support for this
tion and sub-speciation in bacteria can be explained as the re(a)
(b)
sult of macroevolution events
Genetic divergence
Functional speciation
mediated by HGT, an alternative
to fixation of point mutations.
Our proposal stems from the fact
that it is the acquisition of new
Species 2
Species 2
genes that allows a new specific
survival strategy (and thus the
concept of a new species). Further adaptation to the newly conSpecies 1
Species 1
quered niche will occur by successive point mutations, and only
after millions of years will the
1 2 3 4 5 6
1 2 3 4 5 6
new species be recognized by hyTime
Time
bridization or 16S RNA sequenctrends in Microbiology
ing. Thus, we have to distinguish
Fig. 1. A distinction between overall genetic divergence and functional speciation. (a) Genetic diverbetween the radical speciation
gence can be measured by 16S RNA sequencing or DNA–DNA hybridization. A divergence of ≈5% is
event (by HGT) and installation
required for two bacteria to be considered of different species. At time point 1, a bacterium of species
of the new ‘variant’ as a recog1 (light grey dots) acquires virulence determinants by horizontal gene transfer (HGT) (dark grey dots).
nized species (by chromosomal
The dark grey bacterial population undergoes divergent evolution because it colonizes a different
divergence), as shown in Fig. 1.
niche. Only at time point 6 will the dark grey species be considered different from the light grey
HGT in eukaryotes: the lessons
from bacteria
If many bacterial speciation events
can be traced to the acquisition
TRENDS
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species. (b) Functional speciation can be measured by the ability to colonize different ecological
niches. HGT-mediated acquisition of virulence determinants by a bacterium allows colonization of
niche 2. Thus, the dark grey should immediately (time point 1) be considered species 2. The final
result (at time point 6) is the same, but genetic divergence does not tell us why dark grey and light
grey bacteria are evolving differently.
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hypothesis comes from the now widely accepted notion that eukaryotes are, in essence and origin, an assemblage of the components of prokaryotic cells25.
The same HGT mechanisms that produce speciation
in bacteria should, in principle, operate in these assemblages. In fact, massive HGT from endosymbiotic
bacteria (including the ancestors of chloroplasts and
mitochondria) to the nucleus forms the basis of the
theory of the origin of eukaryotes.
It is unlikely that appropriation of bacterial genomes,
or their parts, would occur only once in evolution.
There are strong indications that a very successful series of massive HGT events occurred ~2 billion years
ago, when the earth changed from a reducing to an
oxidizing atmosphere, and if they occurred then, they
will continue to occur forever. We have examples that
conjugation from bacteria to eukaryotes still occurs
nowadays under laboratory conditions (conjugative
transfer to yeast, filamentous fungi and plants) as
well as in natural environments (T-DNA transfer to
plants)6. Furthermore, eukaryotes are naturally transformable (DNA in the form of calcium phosphate
precipitates or liposome complexes can enter most
animal cells by phagocytosis). Intracellular, and even
extracellular, pathogenic bacteria can transfer DNA
to the nucleus of infected cells7,26. Any exogenous
cytoplasmic DNA is picked up by the nucleus and integrates in the chromosomes by illegitimate recombination. Thus, bacterial and viral DNAs are constantly
being integrated in the chromosomes of plants and
animals today, by the known genetic mechanisms
of conjugation, transformation and transduction (in
evolutionary terms, retroviruses and other integrative
viruses are exact parallels of bacteriophages).
It can therefore be concluded that eukaryotes possess the same capacity and similar mechanisms for
effective HGT as prokaryotes do, and laboratory experiments have shown that these mechanisms are
functional. Given the instruments and the opportunities, is it possible that they are not being extensively
used? It is only 98% true that the genomes of humans
and other primates are 98% identical – they are almost 100% identical in almost every gene, and the
process of speciation probably consisted of the acquisition of one or several sets of new genes by HGT. To
mention just a single example, Alu sequences are very
successful transposable elements that entered the ancestral primate germ line ~60 million years ago (by
HGT?). They might have played a pivotal role in the
speciation of primates27,28. Which are the ‘humanspecific’ genes? Are there humanization gene islands?
Our prediction is that they will be found. We would
like to finish with the same phrase used by Ford
Doolittle in a recent review that emphasizes the role
of HGT in genome evolution29 ‘biologists might rejoice
in and explore, rather than regret or attempt to dismiss, the creative evolutionary role of HGT’. We have
attempted to do this here.
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cAMP signalling in pathogenic fungi:
control of dimorphic switching and
pathogenicity
M. Ines Borges-Walmsley and Adrian R. Walmsley
T
he ability of dimorphic
Morphological changes in pathogenic
the response to nutritional
pathogenic fungi to
fungi often underlie the development of
deprivation and, as such, is a
switch between differ- virulence and infection by these organisms. response to an environmental
ent morphological states apOur knowledge of the components of the
stress. Pathogenic fungi appears to be an important virucell signalling pathways controlling
pear to have adapted related
lence determinant as mutant
morphological switching has, to a large
cell signalling pathways to
strains lacking this ability extent, come from studies of pseudohyphal control morphological switchoften have reduced virulence
growth of the model organism
ing during infection. Here, we
or are avirulent. The genes
Saccharomyces cerevisiae, in which
review our current knowledge
controlling
morphogenesis
control is exerted via changes in the
of the cAMP signalling pathhave therefore been the focus intracellular cAMP and mitogen-activated way in S. cerevisiae, drawing
of many investigations, as they protein kinase cascades. There is evidence parallels with pathogenic fungi.
have great potential as targets
that pathogenic fungi also utilize these
for novel antifungal drugs.
pathways to control dimorphic switching
Morphological switching in
Most of our knowledge of the
between saprobic and pathogenic forms
S. cerevisiae
signalling pathways involved
and, as such, the elements of these
Diploid cells of the budding
in controlling morphological
pathways have potential as drug targets.
yeast S. cerevisiae can be inswitching has been provided
duced to produce pseudoby studies of the switch from M.I. Borges-Walmsley and A.R. Walmsley* are in the hyphae under conditions of
Divn of Infection and Immunity, Institute of
yeast to pseudohyphal growth
nitrogen starvation. This morBiomedical and Life Sciences, University of Glasgow,
in the model organism Sacphological transition entails
Glasgow, UK G12 8QQ.
charomyces cerevisiae. These
changes in cell morphology,
*tel: 144 141 330 3750,
fax: 144 141 330 3751,
studies have revealed that the
budding pattern and cell separe-mail: [email protected]
signalling pathways are conation: the cells switch from an
trolled by both cAMP and
ovoid to an elongated shape;
mitogen-activated protein kinase (MAPK) signal they bud apically and the buds remain attached, retransduction pathways. It has become clear that sulting in chains of cells that constitute the pseudopseudohyphal growth in S. cerevisiae is one aspect of hyphae. In vitro, they also acquire the ability to invade
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TRENDS
IN
MICROBIOLOGY
133
VOL. 8
NO. 3
PII: S0966-842X(00)01698-X
MARCH 2000